Methods and systems useful for foodborne pathogen detection

ABSTRACT

Described are methods and systems useful in the processing of food samples to detect pathogenic cells in the samples. Embodiments include homogenizing a food sample, digesting solids of the homogenized food sample, and removing lipids from the homogenized food sample. Cells in the sample can then be concentrated using hollow fiber membrane filtration. Regeneration cycles can enable multiple reuses of the hollow fiber membrane filter. Systems and processes herein can be automated, under the control of a computer.

RELATED APPLICATION

This application is a continuation of and claims the benefit of priorityof International Application Serial No. PCT/US2013/034349, filed Mar.28, 2013, which claims the benefit of priority of U.S. ProvisionalPatent Application Ser. No. 61/617,036 filed Mar. 28, 2012, each ofwhich is hereby incorporated by reference in its entirety.

GOVERNMENT INTEREST

This invention was made with government support under contract number59-1935-8-850 awarded by the United States Department of Agriculture.The government has certain rights in the invention.

FIELD OF THE INVENTION

In certain aspects, the present invention relates to methods and systemsthat are useful for detecting the presence of pathogenic cells in a foodsample, and in particular embodiments to methods and systems forprocessing solid food samples to concentrated, assayable liquid samples.

BACKGROUND

Pathogenic bacteria in foods are the cause of 90% of the cases ofreported foodborne illnesses. The Centers for Disease Control andPrevention estimate that there 76 million cases of foodborne illnesseseach year in the United States, resulting in hospitalization of 325,000people, 5,500 deaths, and an annular cost of $7 to $23 billion. E. coliO157:H7, Salmonella and Listeria monocytogenes are the pathogens ofmajor concern. Ground meat containing E. coli O157:H7 is now consideredto be an adulterated food while Listeria monocytogenes has emerged asone of the most important food pathogens with a “zero tolerance”criterion for it in ready-to-eat processed (lunch) meats and dairyfoods.

The genus Listeria is comprised of six species, L. monocytogenes, L.ivanovii, L. seeligeri, L. innocua, L. welshimeri, and L. grayi. Ofthese species, only L. monocytogenes is harmful to humans. Consumptionof contaminated food may cause meningitis, encephalitis, liver abscess,headache, fever and gastroenteritis (diarrhea) in immunologicallychallenged individuals and abortion in pregnant women. L. monocytogenesis ubiquitous in nature and can be found in meat, poultry, seafood, andvegetables. Occurrence of this organism could be as high as 32%. In afood sample, L. monocytogenes is often present in close association withother nonpathogenic Listeria species, thereby complicating the specificdetection procedures. A successful detection method ideally detects onlyL. monocytogenes in the presence of larger populations of nonpathogenicListeria and other background resident bacteria.

The food processing industry annually carries out more than 144 millionmicrobial tests costing $5 to $10 each. About 24 million of these testsare for detection of food pathogens based on biochemical profileanalysis, immunogenic tests (such as enzyme linked immuno-sorbent assaysor ELISA), and DNA/RNA probes. These tests are reliable but most requiretwo to seven days to complete because of the steps that are needed toresuscitate cells, increase cell numbers or amplify genetic materialneeded for detection. This time period is too long for real-timedetection of contamination in a food plant and is sufficiently long forcontaminated food to be formulated, processed, packaged, shipped, andpurchased and eaten by the consumer. Current tests require at leastseveral days to confirm presence of Listeria monocytogenes. The numberof annual tests is only expected to increase due to heightened consumerconcerns about food safety and the requirement of compulsory testing.

In general, diagnostic tools used for detecting or quantitatingbiological analytes rely on ligand-specific binding between a ligand anda receptor. Ligand/receptor binding pairs used commonly in diagnosticsinclude antigen-antibody, hormone-receptor, drug-receptor, cell surfaceantigen-lectin, biotin-avidin, substrate/enzyme, and complementarynucleic acid strands. The analyte to be detected may be either member ofthe binding pair; alternatively, the analyte may be a ligand analog thatcompetes with the ligand for binding to the complement receptor.

A variety of devices for detecting ligand/receptor interactions areknown. The most basic of these are purely chemical/enzymatic assays inwhich the presence or amount of analyte is detected by measuring orquantitating a detectable reaction product, such as a detectable markeror reporter molecule or ligand. Ligand/receptor interactions can also bedetected and quantitated by radiolabel assays.

Quantitative binding assays of this type involve two separatecomponents: a reaction substrate, e.g., a solid-phase test strip and aseparate reader or detector device, such as a scintillation counter orspectrophotometer. The substrate is generally unsuited to multipleassays, or to miniaturization, for handling multiple analyte assays froma small amount of body-fluid sample.

In recent years, there has been a merger of microelectronics andbiological sciences to develop what are called “biochips.” The term“biochip” has been used in various contexts but can be defined as a“micro fabricated device that is used for delivery, processing, andanalysis of biological species (molecules, cells, etc.).” Such deviceshave been used, among other things, for the direct interrogation of theelectric properties and behavior of cells (Borkholder et al. “PlanarElectrode Array Systems for Neural Recording and ImpedanceMeasurements”, IEEE Journal of Microelectromechanical Systems, vol 8(1),pp. 50-57, 1999); impedance-based detection of protein binding tosurfaces, antigen-antibody binding, and DNA hybridization (DeSilva etal., “Impedance Based Sensing of the Specific Binding ReactionStaphylococcus Enterotoxin B and its Antibody on an Ultra-thin PlatinumFilm,” Biosensors & Bioelectronics, vol. B 44, pp 578-584, 1995);micro-scale capillary electrophoresis (Wooley et al., Ultra High SpeedDNA Sequencing Using Capillary Electrophoresis Chips,” AnalyticalChemistry, vol. 67(20), pp. 3676-3680, 1995); and optical detection ofDNA hybridization using fluorescence signals in the commerciallyavailable “DNA-chips” (Fodor et al., “Light-directed SpatiallyAddressable Parallel Chemical Synthesis,” Science, vol. 251, pp.767-773).

One of the most interesting uses of biochips is for the detection ofsmall quantities of pathogenic bacteria or toxigenic substances in food,bodily fluids, tissue samples, soil, etc. In applications such as thescreening of food products for the presence of pathogenic bacteria, itwould be beneficial to detect between 100 and 1000 microorganisms permilliliter of sample, with a sample volume of a couple of milliliters.Not counting the fact that bacteria are substantially larger than singlebiomolecules (about 2 micrometers vs. about 10-100 Angstroms), 1000cells are approximately equivalent to a 10⁻⁵ femto-moles of cells, whichgives an idea of the difficulty in directly detecting such a smallnumber suspended in a volume of 1 or 2 ml, along with large numbers offood debris, proteins, carbohydrates, oils, and other bacteria.Additionally, in many cases the screening technique must be able todiscern between viable and dead cells. Many bacteria will not producetoxins when not viable and consequently will not be pathogenic in thatstate. DNA detection methods, which search for DNA sequences specific tothe pathogen of interest, can be extremely sensitive because they relyon the very specific binding of complementary DNA strands, often coupledwith Polymerase Chain Reaction (PCR) for amplification. But the detectedDNA fragments cannot reveal whether the pathogen was viable or not.These are the main reasons why current methods of detection almostalways involve a growth step, in which the bacteria are cultured toincrease their numbers by several orders of magnitude. Once the bacteriaare amplified to a large number, visual detection of colonies orEnzyme-Linked Immunosorbent Assays (ELISA) confirm their presence in theoriginal sample. Even though bacteria can multiply very rapidly, thisamplification by means of extended growth makes conventional detectionmethods extremely lengthy, taking anywhere from 2 to 7 days. Thus, goalsin industry have been to reduce the time of sample processing andanalysis, while also retaining or heightening the sensitivity of theanalysis.

SUMMARY

In one aspect, the present invention provides a method for processing afood sample. The method includes preparing a liquefied extract of a foodsample, and processing the liquefied extract by hollow fiber membranefiltration to form a concentrate of cells from the liquefied extract.The step of preparing preferably includes enzymatically digesting thefood sample. The hollow fiber membrane filtration can be conducted undercross flow conditions, with flow of a liquid on the permeate side of thehollow fiber(s). The method can also include assessing the concentrateof cells for the presence of pathogenic cells.

In another aspect, the present invention provides a method for preparinga liquefied extract of a food sample. The method includes homogenizing asolid food sample under controlled shear conditions to form ahomogenized food sample substantially retaining a viable cellularcontent of the solid food sample. The method further includesenzymatically digesting the homogenized food sample to hydrolyzeproteins in the homogenized food sample and form a digested food sample.Additionally, the digested food sample is processed to remove lipidsfrom the digested food sample, to form a lipid-depleted food sample. Thelipid-depleted food sample can be processed by hollow fiber membranefiltration, desirably under cross flow conditions, to concentrate cellsin a retentate and generate a permeate. The method can also includeassessing the concentrated cells for the presence of pathogenic cells.

In another aspect, the present invention provides a method for testingfor pathogenic cells. The method includes contacting a viable cellularconcentrate from a food sample with a fluorescent antibody dye thatspecifically binds to the pathogenic cells, to form a contacted sample.The method further includes processing the contacted sample underconditions to remove unbound amounts of the fluorescent antibody dye, toform a dye-depleted sample, and spectroscopically interrogating thedye-depleted sample for the fluorescent antibody dye. The viablecellular concentrate can be prepared by a process including:homogenizing a solid food sample under controlled shear conditions toform a homogenized food sample substantially retaining a viable cellularcontent of the solid food sample; enzymatically digesting thehomogenized food sample to hydrolyze proteins in the homogenized foodsample and form a digested food sample; processing the digested foodsample to remove lipids from the digested food sample, to form alipid-depleted food sample; and concentrating the lipid-depleted foodsample in cells.

In another aspect, the invention provides a method for processing ahollow fiber membrane that has contacted a liquefied food sample. Themethod includes (i) passing a sodium hydroxide solution through thehollow fiber membrane; (ii) passing an aqueous medium through the hollowfiber membrane after step (i) to rinse sodium hydroxide residues fromsaid membrane; (iii) passing a sterilant solution through the hollowfiber membrane after step (ii); and

(iv) passing an aqueous rinse medium through the hollow fiber membraneafter step (iii).

In another aspect, the invention provides a method for concentrating andrecovering cells from a food sample. The method includes (i) passing aliquefied extract of the food sample through the interior of a hollowfiber membrane to form a permeate exterior of the hollow fiber membraneand a liquid concentrate of cells in the interior of the hollow fibermembrane; and (ii) passing an aqueous solution against an exteriorsurface of the hollow fiber membrane during said passing step (i). Thepassing step (ii) can include passing the aqueous solution co-current toa flow of the liquefied extract through the interior of the hollow fibermembrane. The liquefied extract can be prepared by a process includingenzymatic digestion of the food sample.

In another aspect, the invention provides a method for assessing a solidfood sample for the presence of pathogenic cells. The method includes(i) suspending the solid food sample in an aqueous medium to form asuspended sample medium; (ii) homogenizing the suspended sample mediumunder controlled shear conditions to form a homogenized food sample,said homogenizing conducted under conditions effective to substantiallyretain any viable pathogenic cells from the solid food sample; (iii)enzymatically digesting the homogenized food sample to hydrolyzeproteins in the homogenized food sample and form a digested food samplemedium; (iv) processing the digested food sample medium to remove lipidsand form a lipid-depleted food sample extract liquid; (v) passing thelipid-depleted food sample extract liquid through the interior of ahollow fiber membrane to form a permeate exterior of the hollow fibermembrane and a liquid concentrate containing cells in the interior ofthe hollow fiber membrane; (vi) passing an aqueous solution against anexterior surface of the hollow fiber membrane during said passing step(v); and (vii) assessing the liquid concentrate containing cells for thepresence of viable pathogenic cells. The method can also include, afterstep (vii): (viii) passing a defouling liquid through the hollow fibermembrane; (ix) passing an aqueous rinse medium through the hollow fibermembrane to remove defouling solution residues from step (viii); (x)passing a sterilant liquid through the hollow fiber membrane; and (xi)passing an aqueous rinse medium through the hollow fiber membrane afterstep (x) to remove sterilant liquid residues from the hollow fibermembrane. The process can also include repeating steps (i) to (vii)after step (xi).

In additional embodiments, methods as described above or elsewhereherein can be conducted wherein any one, any combination, or all of therecited steps are conducted under the control of a controller, such as acomputer.

Still further embodiments of the invention provide systems orinstruments for processing food sample material, which are configured tocarry out steps as in any method described herein.

Additional embodiments of the invention, as well as features andadvantages thereof, will be apparent from the descriptions herein.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is a flow chart of one embodiment of a method of the invention.

FIG. 2 provides a schematic diagram of one embodiment of a system andmethod for processing a food sample in accordance with the invention.

FIG. 3 is an illustration of a display for one computer-controlledsystem and method of the invention.

FIGS. 4 to 8 show results obtained using an embodiment of a system andmethod of the invention.

DETAILED DESCRIPTION

For the purpose of promoting an understanding of the principles of theinvention, reference will now be made to embodiments, some of which areillustrated in the drawings, and specific language will be used todescribe the same. It will nevertheless be understood that no limitationof the scope of the invention is thereby intended. Any alterations andfurther modifications in the described embodiments, and any furtherapplications of the principles of the invention as described herein arecontemplated as would normally occur to one skilled in the art to whichthe invention relates.

As disclosed above, in certain aspects the present invention relates tomethods and systems for processing food materials that are useful forthe detection of pathogenic cells in the materials. In the discussionsthat follow, a number of potential features of reagents, food samples,system equipment, steps, physical or chemical conditions, or otheraspects, are disclosed. It is to be understood that each such disclosedfeature or features can be combined with the features discussed in theSummary above or recited in the Specific Listed Embodiments below, toform a disclosed embodiment of the present invention. As well, it willbe understood by those skilled in the art that methods and systemsdescribed herein will with preference be able to concentrate and detectviable pathogenic cells from food samples. Accordingly, liquid and otherreagents or materials to contact the sample, e.g. feeds to systemsdescribed herein, are sterile as appropriate to avoid contaminating thesample with extraneous viable cells.

With reference to FIG. 1, shown is a flow chart illustrating certainmethod embodiments of the invention, for processing a food sampleusefully for detection of a pathogen, such as a pathogenic cell, in thesample. A first step or operation 10 includes sampling of the food,which is typically a solid food, and preparing a homogenate of the foodsample. This can be accomplished in any suitable manner. In onepreferred mode, the food sample is processed using a Seward Stomacherlab blender (Thetford, Norfolk, UK). The STOMACHER lab blender has twopaddles and a baffle which serve to shape the food into a ring and use acombination of stirring and crushing to homogenize the food. Thistechnique has been shown to release more deep-seated microbes than otherfood safety sample preparation methods. This method is of practicalapplication to food pathogen research because it provides a way ofdisrupting bacterial interactions on and in food surfaces while allowingcells to remain viable. Use of a STOMACHER lab blender is listed as astandard method in the USDA/Food Safety Inspection Service (FSIS)Microbiology Laboratory Guidebook, Chapter 3, Section 3.31 “foodhomogenates.” Other suitable homogenization methods, however, may alsobe used. The illustrative method also includes a step 12 to pretreat thefood homogenate to modify its content. For example, the pretreatment maybe effective to further liquefy the food sample. In preferred modes, thepretreatment will involve a digestion of food solids remaining in thehomogenate, and in one particularly beneficial mode an enzymaticdigestion. The enzyme and conditions used will depend upon the type offood sampled, and other particular factors. A number of illustrativedigestive enzymes are identified in the discussions below. After suchpretreatment a step 14 of pre-filtration of the homogenate can beconducted. Such prefiltration will also modify the content of thehomogenate, for example by removal of potential membrane foulants, suchas lipids. The homogenate is then subjected to a hollow membranefiltration step 16, which is desirably conducted in a cross flowpattern. The filtration step 16 uses a membrane filter effective toretain (not pass) cells. A number of hollow fiber membranes suitable forthis purpose are commercially available, and can be used. They willtypically have a pore size that is less than the diameter of the cellsin the sample to be concentrated for analysis. The filtration step 16will typically concentrate the food sample medium by many fold, forexample at least 100 fold. Higher fold concentrations are also possibleand useful in many instances, for example at least 500 fold, and oftenin the range of 500 fold to 1000 fold. The concentrated sample after thefiltration step 16 can then be further processed in step 18, in a mannereffective to detect the presence of any target pathogens, and especiallypathogenic cells, in the concentrated sample. A number of pathogenspecific detection methods are known and can be used. These may include,for example, microfluidic detection methods (e.g. microfluidic chips),immunoassays, polymerase chain reaction methods, bacteriophage reportermethods, Raman spectroscopic methods, and/or light scattering methods.After processing a food sample homogenate for concentration ofpathogens, the hollow fiber membrane filter used in step 16 can beregenerated in step 20. This regeneration preferably removes foulantsfrom the membrane surfaces, for example using a liquid substance such asan aqueous sodium hydroxide solution to remove cell membranes and otherdebris, and a sterilant such as an alcohol or aqueous alcohol solutionto sanitize the membranes and thereby kill viable cells remainingthereon from the previous filtration run. The hollow membrane filterunit can then be used in a subsequent, similar operation to process asubsequent food sample homogenate. In experimental protocol, the processcharted in FIG. 1 may also include a sample spike step 22, e.g.immediately prior to the hollow fiber membrane filtration step 16, inwhich a pathogen to be tested in the method or associated system isadded to the sample.

With reference now to FIG. 2, shown is a schematic diagram that usefullyillustrates certain method, system and instrument embodiments of thepresent invention. While some discussions that follow focus upon systemelements in which method steps are or can be performed, it will beunderstood that these descriptions likewise disclose inventive methodsand instruments, which can be conducted in the illustrated system, orotherwise. Shown in FIG. 1 is system 30 useful for processing a foodsample to detect the presence of one or more pathogenic cells, such aspathogenic bacteria, in the food sample. System 30 includes a hollowfiber membrane unit or module 32 and a pump 34, preferably a peristalticpump, fluidly coupled thereto. Such a peristaltic pump may, for example,be capable of providing a flow rate of 0.004 to 50 mL per minute, as inan Ismatec compact analog pump. System 30 also includes, in fluidcommunication with pump 34, a sample reservoir 36, a concentrated samplerecovery buffer reservoir 38, a defoulant liquid reservoir 40 (e.g.sodium hydroxide solution or another caustic), and a sterilant reservoir42. The reservoirs 36, 38, 40, and 42 feed through respective flowconduits 36 a, 38 a, 40 a and 42 a, to a four-to-one selector valve 44,which in turn feeds through flow conduit 44 a to pump 34. In thisfashion, valve 44 can be actuated to selectively direct fluid from anyone of reservoirs 36, 38, 40 and 42 to pump 34. The above mentioned flowconduits, and other flow lines or conduits mentioned herein, can beprovided by suitable polymeric tubing. Pump 34 feeds through pressuretransducer 46 (for example allowing a maximum pressure of 60 psi) tohollow fiber filtration membrane module 32, and in particular to inlet32 a thereof that feeds into the interior of the hollow fibermembrane(s) of the module 32. Outlet 32 b of module 32 feeds from theoutlets of the hollow fiber membrane(s) of module 32 to a two-way valve50, operable to selectively direct flow to either a retentate returnline 52 or a sample collection line 70. Retentate return line 52 feedsback to sample reservoir 36. In this manner, sample can be passed underthe power of pump 34 in a continuous loop that includes membrane module32, and concentrated over time by repeated passages through module 32.

System 30 also includes a second pump 54, such as a peristaltic pump asdescribed above, and a permeate-side fluid reservoir 56 (for exampledistilled water) fluidly coupled to pump 54 by flow conduit 56 a. Pump54 feeds through feed conduit 54 a to pressure transducer 58 (e.g.allowing a maximum pressure of 60 psi), which in turn feeds through flowconduit 60 to permeate-side inlet 32 c of membrane module 32.Permeate-side opening 32 c feeds into a space within a chamber of module32 and external of the hollow fiber membrane(s) that extend throughmodule generally from inlet 32 a to outlet 32 b. Permeate-side outlet 32d feeds from such chamber into a flow conduit 64 which in turn feeds topermeate collection reservoir 68. A first flow meter 62 is operablyassociated with flow conduit 60 and a second flow meter is operablyassociated with flow conduit 64. In this manner, the rate of flow intoand out of the permeate side of module 32 can be measured and a visiblereadout provided to a user, for example using a computer-controlledsystem with a display (further described below).

System 30 includes a sample concentrate collection line 70 fluidlycoupled to two-way valve 50, which in turn feeds to a sampleconcentration collection reservoir 72. System 30 also includes a liquidlevel sensor 74 operably associated with sample reservoir 36. Sensor 74can be operable to sense when the liquid sample in reservoir 74 falls toor below a predetermined level. System 30 further includes a two-wayvalve 76 in retentate return flow conduit 52, fluidly coupled to flowconduit 78 which feeds into a sterilant reservoir 80 (e.g. a bleachsolution such as a 10% bleach solution). A turbidity sensor 82 isoperably associated with flow conduit 64 that receives flow from thepermeate-side outlet 32 d of module 32. Turbidity sensor 82 can beoperable to sense turbidity in the permeate fluid exiting outlet 32 d,which when sensed can provide a signal to actuate valve 76 to stop flowthrough retentate return flow conduit 53 back to sample reservoir 36 andinstead direct liquid flow to flow conduit 78 and into sterilantreservoir 80. In this fashion, the turbidity sensed by sensor 82 can beindicative of a membrane breach in module 32, and the contaminatedpermeate can be redirected to the sterilant reservoir 80 to kill anypathogenic cells in the contaminated permeate. In addition to or as analternative to providing permeate-side liquid from reservoir 56, apermeate return flow conduit 84 can be provided and fluidly coupled toflow conduit 56 in turn feeding to pump 54. In this manner, permeateliquid can be recirculated in a loop through the permeate side of module32 under the power of pump 54.

In operation, a food sample is provided to sample reservoir 36. Such afood sample may be, and preferably is, a liquid sample that has beenpre-processed as in steps 10, 12 and 14 discussed in conjunction withFIG. 1. With valve 44 set to feed liquid from sample reservoir 36 topump 34, and with valves 50 and 76 set to feed retentate back to samplereservoir 36, pump 34 is operated to pump fluid through the flow loopincluding module 32 and thereby pass the sample liquid through theinterior of the hollow fiber membrane(s) of module 32. At the same time,pump 54 is operated to pump permeate-side fluid, for example aphysiologically-acceptable buffer such as a phosphate buffer, fromreservoir 56 (and/or recirculated permeate fluid from flow conduit 84)through the permeate side of module 32, from inlet 32 c to outlet 32 d.A cross-flow filtration is thereby established in module 32, with apositive pressure gradient (transmembrane pressure) maintained from theinterior of the hollow fiber(s) of module 32 to the exterior thereof(permeate side). The operation of pumps 34 and 54 is continued, therebycreating an increasingly concentrated retentate that retains any viabletarget pathogenic cells. When the retentate is sufficientlyconcentrated, for example as can be signaled by liquid level sensor 74,valve 50 can be switched to direct flow to flow conduit 70 and intoconcentrated sample collection reservoir 72. After sample reservoir 36is emptied of sample, valve 44 can be actuated to direct an amount ofrecovery buffer from reservoir 38 through the hollow fiber membrane(s)of module 32 to collect any remaining cells within the associated flowpath (e.g. adhered to the interior surface of the hollow fibermembrane(s)) and deliver them through flow conduit 70 and intocollection reservoir 72. The amount of recovery buffer fed in thisoperation can be carefully measured to avoid substantial, dilution ofthe sample liquid as it enters collection reservoir 72. In this regard,the volume of recovery buffer fed in such an operation can be a fractionof the volume of the food sample liquid initially loaded in samplereservoir 36, for example with a recovery buffer:initial sample volumeratio of 1 to 5 or greater, more preferably 1 to 10 or greater, and evenmore preferably 1 to 20 or greater. In one embodiment, such ratio is 1to 25. An aqueous surfactant solution such as an aqueous polysorbatesolution is preferred as a recovery buffer. Such polysorbate or othernonionic or ionic surfactant solutions are desirably relatively dilutesolutions, for example less than about 0.01% v/v, more desirably lessthan 0.005% v/v, to avoid significant foaming in use in system 30.Aqueous polysorbate or other surfactant concentrations as low as about0.001% can be used for these purposes in some embodiments. Polysorbate20 (e.g. as provided commercially as Tween 20) is a preferredsurfactant. The surfactant solution is desirablyphysiologically-acceptable so that it does not significantly impact theviability of cells in the collected retentate sample. Optionally, duringprocessing-through of the recovery buffer, flow of water or anothersuitable liquid, typically aqueous, on the permeate side may bemaintained with a positive interior-to-exterior transmembrane pressuregradient also maintained. In another mode, no liquid flow on thepermeate side is used during passage of the recovery buffer.

After recovery of the concentrated retentate sample in reservoir 72, andprior to processing another food sample through system 30, a systemregeneration cycle can be conducted. For these purposes, valve 44 can beactuated to direct flow from defoulant liquid (e.g. sodium hydroxidesolution) from reservoir 40 through the hollow fiber membrane(s) ofmodule 32, so as to remove cell membranes and/or other foulant materialsfrom the surfaces of the hollow fiber membrane(s) of module 32. Duringprocessing-through of the defoulant liquid, flow of water or anothersuitable liquid, typically aqueous, on the permeate side may bemaintained with a positive interior-to-exterior transmembrane pressuregradient also maintained. Amounts of the defoulant liquid passed throughthe membrane(s) of module 32 can be collected in reservoir 68.

After passage of the defoulant liquid, remaining residues of thedefoulant liquid can be removed by passing an aqueous rinse medium,acceptably distilled water, through the hollow fiber membrane(s) ofmodule 32 and the flow paths leading to and from such membrane(s).During processing-through of the aqueous rinse medium, flow of water oranother suitable liquid, typically aqueous, on the permeate side may bemaintained with a positive interior-to-exterior transmembrane pressuregradient also maintained. Amounts of the aqueous rinse medium and rinseresidues passed through the membrane(s) of module 32 can be collected inreservoir 68.

After rinsing the module 32 as noted above, valve 44 can be actuated todirect a sterilant liquid from reservoir 42 through the interior ofhollow fiber membrane(s) of module 32 and through the flow paths feedingto and from module 32, to kill any viable cells remaining therein.Again, during processing-through of the sterilant liquid medium, flow ofwater or another suitable liquid, typically aqueous, on the permeateside may be maintained with a positive interior-to-exteriortransmembrane pressure gradient also maintained. Amounts of thesterilant liquid medium passed through the membrane(s) of module 32 canbe collected in reservoir 68.

After passage of the sterilant liquid, remaining residues thereof can beremoved by passing an aqueous rinse medium, acceptably distilled water,through the hollow fiber membranes of module 32. A flow of water oranother suitable liquid, typically aqueous, on the permeate side may bemaintained during processing-through of the aqueous rinse medium, with apositive interior-to-exterior transmembrane pressure gradient alsomaintained. Amounts of the rinse medium and rinsed sterilant liquidresidues can be passed through the membrane(s) of module 32 can becollected in reservoir 68.

After the regeneration cycle including the above defoulant, rinse,sterilization, and rinse steps, the system 30 can be used to processanother food sample charged to reservoir 36. For these purposes, thevarious other reservoirs of system 30 can be sufficiently large forprocessing of multiple samples, or these reservoirs can be recharged aswell.

During processing, pressure on the retentate side can be measured usingpressure transducer 46, and pressure on the permeate side can bemonitored using pressure transducer 58. Flow into permeate-side inlet 32c can be monitored using flow meter 62, and flow out of permeate-sideoutlet can be monitored using flow meter 66. These pressure and flowreadings can be used to maintain or adjust the speed of pumps 34 and 54to maintain the flow and pressure conditions necessary for thefiltration with module 34.

Should a membrane breach occur, turbidity monitor 82 will detect anincrease in turbidity. This can provide a signal to actuate valve 76 toredirect flow of the retentate in flow conduit 52 from the samplereservoir 36 to instead the bleach, caustic or other sterilant reservoir80 to kill any pathogenic cells in the permeate. The increase inturbidity may also provide a signal to turn off pumps 34 and 54, eitherimmediately or after a predetermined period of time for shunting ofcontaminated permeate to reservoir 80.

The preferred system 30 also includes a controller 100, such as acomputer, that automatically controls, and/or enables user inputs tocontrol, some or all aspects of operation of system 30 as discussedabove. For these purposes, controller 100 can be configured toelectronically communicate with system 30 components so as to have thecapacity to receive and/or send signals to the components (e.g. pumps,valves, sensors, or meters as discussed above) to achieve the notedoperations, and potentially others. Controller 100 can also becommunicably associated with a user input device, such as a keyboard102, and/or a display device 104 which may also serve as a touch screeninput in some embodiments. Computer or other controller 100 can operatesoftware that enables display and control functions as noted herein forsystem 30. In one embodiment, software commercially available as LabVIEW2009f3, available from National Instruments, is utilized. In thisregard, FIG. 3 shows a suitable display for system 30 generated by thisLabVIEW 2009f3 software.

The membrane module 32 can contain any suitable number of hollow fibermembranes, and typically a plurality of membranes will be used in abundle. For example, 3 or more, 5 or more, or 10 or more hollow fibermembranes can be used. In one embodiment, 12 hollow fiber membranes areused. The membranes can be made from any suitable material. Polysulfonehollow fiber membranes are suitable. The membranes can have a pore sizesufficient to retain (prevent passage) of cells. A pore size of about0.2 micrometers is suitable for this purpose. The inner diameter of themembranes may suitably be about 100-300 micrometers, for instance 280micrometers, and an outer diameter may suitably be 150 to 400micrometers, for instance about 360 micrometers. The membrane thicknessmay suitably be about 50 to 100 micrometers, and typically about 80micrometers. In one module embodiment, about 0.2 micrometer pore sizehollow fiber membranes having an inner diameter of 280 micrometers, anouter diameter of 360 micrometers, and a length of about 27 centimeters,are bundled together. 12 such hollow fibers can be used. The fiberbundle is packed into polymeric tubing, for example polyether etherketone (PEEK) tubing, having an inner diameter of about 2 millimetersand an outer diameter of about 3.2 millimeters. Each end of the moduleis then sealed with two layers of epoxy to prevent leakage, and toseparate the retentate side from the permeate side. “T” connectors areprovided on the tubing and around the fiber bundle to provide apermeate-side inlet and a permeate-side outlet.

The concentrated sample provided by system 30 can be processed in anymanner desired to assess for target pathogenic cells. Any of thosemethods, and systems or devices therefore, described hereinabove may beused. In further embodiments, such detection systems or devices can beoperably associated with and part of system 30, receiving feed of thenecessary sample for detection from reservoir 72 or potentially directlyfrom concentrated sample collection conduit 70. Also in theseembodiments, the feed to and operation of the detector may be controlledby controller 100.

In work to date, a working embodiment of system 30 has been used inseveral modes of testing, results of which are shown in FIGS. 4-8.Generally in this testing, a cell concentration (CC) mode and a cellrecovery (CR) mode were used. The micro-flora was concentrated under theCC mode, during which the initial sample was feed through the membranemodule and the microorganisms that were rejected by the membrane wereconcentrated in the retentate. The retentate was returned to the samplereservoir, while the permeate was collected (to avoid microbialcontamination) and disposed later. The initial CC mode operation wasfollowed by an elution phase under CC mode using 10 mL deionized wateror PBS supplemented with 0.002% (v/v) tween 20 per 250 mL of sampleinitially applied. The CC mode was run with the speeds of pump 34 andpump 54 adjusted respectively to 100% and 20% of their maximum, giving alaminar cross-flow velocity of 1.126 m s-1 (corresponding to a Reynoldsnumber of 351). The cell recovery was carried out under the CR mode,during which the retentate was diverted into the sample collectionreservoir instead of being circulated back to the sample vessel. Underthe CR mode, only pump 34 was powered and set at 100% of its maximumspeed. The switch between CC and CR mode was accomplished by appropriatecontrol of the valves. Baseline conditions were established by runningsterile deionized water through the system. The sample load was 250 mLof homogenate (made from 25 g chicken flesh and skin) per run. All theexperiments were carried out at room temperature.

Provided in FIG. 4 is a table showing the concentration factors andpercentage recoveries for the Salmonella-spiked chicken rinse foodsample pre-processed before the hollow fiber membrane filtration asshown and described in connection with FIG. 1. As can be seen, highconcentration factors and high percentages of recovery of the spikedorganism were achieved. FIG. 5 shows colony growth from theSalmonella-spiked food samples concentrated in system 30 as describedimmediately above vs. non-concentrated, when plated on selective ornon-selective growth media. As can be seen, colony growth for theconcentrated samples was dramatically higher. In a similar vein, FIG. 6shows a table of the concentration and recovery of natural flora fromthe food sample processed in system 30. Again, high concentrationfactors and good percentage recoveries were achieved.

FIG. 7 shows the results of plating a buffer medium passed throughmodule 32 of system 30 after running a Salmonella-spiked sample asdiscussed above, after an NaOH defouling+rinse operation, and after anNaOH defouling+rinse+70% aqueous ethyl alcohol+rinse operation. As canbe seen, after the NaOH defouling+rinse operation only, many colonieswere formed on the growth medium, whereas after the NaOHdefouling+rinse+70% aqueous ethyl alcohol+rinse operation, no colonieswere formed.

FIG. 8 shows the results of re-use of the membrane module 32 of system30 multiple times under conditions specified above for otherexperimental runs, with the NaOH defouling+rinse+70% aqueous ethylalcohol+rinse operation conducted between each run. As shown, thetransmembrane pressure for the module 32 remained relatively steady in ausable range over many uses.

In a further embodiment, system 30 can include an early detectionfeature for rapid detection of the presence of target pathogenic cellsin the retentate. For these purposes, system 30 can include a dyereservoir 90, for example containing a fluorescent dye that specificallylabels one or more target pathogenic cells to be detected. A suitablefluorescent marker for these purposes includes, for example, greenfluorescent protein (GFP), although others are known and can also beused. System 30 can also include a pump 92 for pumping amounts of dyefrom reservoir 90 into retentate flow conduit 52. If needed or desired,a valve could also be provided between pump 92 and conduit 52 toselectively permit or prevent flow to conduit 52. The opening andclosing of such a valve would be coordinated with the operation of pump92 to feed amounts of the dye to conduit 52 at the appropriate time(s).System 30 can also include a detector 94 associated with retentate flowconduit 52 and operable to detect the presence of the dye in theretentate. In operation, an amount of the dye can be charged to theconduit 52 during a retentate circulation/concentration operation asdescribed above. The dye will specifically bind to target pathogeniccell(s) in the retentate, and some amounts of the dye will remainunbound (free) in solution. Unbound amounts of dye will be continuouslypassed to the permeate in module 32, and thus the signal detected bydetector 94 will diminish over time after input of a measured charge ofdye into the retentate. A remaining reduced, but steady signal wouldindicate the presence of pathogenic cells in the retentate, and providean early indication that the food sample was contaminated with thepathogenic cells. Several such pulses of dye can be added to theretentate over time as the sample continues to concentrate. Eventually,if the target pathogenic cells are present, the remaining signal fromdetector 94 (after removal of the unbound dye through the permeate) willbe strong enough, due to the concentration of the pathogenic cells, toreliably indicate the presence of the target pathogenic cells in theretentate. Detector 94 is preferably a spectroscopic detector thatspectroscopically interrogates the retentate in-line as it flows withinsystem 30. The dye for these purposes is preferably a fluorescent dye,for example a fluorescent antibody dye, that binds specifically to asurface protein on the target pathogenic cell(s). As well, a mixture ofsuch specific dyes could be used in the pulse(s) into the retentate, toenable the detection of different types of pathogenic cells byspecifically binding of a dye to each of the cell types. In such casesthe mixed dyes could each carry the same fluorescent marker and thusprovide a signal additive of the different types of pathogenic cells, orcould each carry a different fluorescent marker and thus provide adistinct signal relative to each of the different types of cellsseparately. The detector 94 could be configured to detect each suchdistinct signal, or one or more additional separate detectors could beincorporated into system 30 to detect the distinct signals. These andother variations will be apparent to those of ordinary skill in the artfrom the descriptions herein.

Target pathogenic cells for detection may be any pathogenic cells ofinterest. The pathogenic cells may in certain embodiments be E. coliO157:H7, Salmonella or Listeria monocytogenes pathogens.

The food sample processed may be any suitable food sample, preferably asolid material. It may be a meat product such as poultry or beef, forexample a ground meat potentially containing E. coli O157:H7, or aready-to-eat processed meat or dairy food that may contain Listeriamonocytogenes, or a meat such as chicken that may contain a Salmonellapathogen. The food sample may also be a dry food such as a cereal orother starch, or a vegetable, in certain embodiments.

The following Examples describe various embodiments of the presentinvention, as well as features and further details that can be used inthe above-described embodiments. Additionally, U.S. Pat. Nos. 7,306,924and 7,547,526, which are incorporated herein by reference, describeadditional details of pathogens, food products, processing conditions,and detection methods and devices that may be utilized in embodiments ofthe present invention.

Example 1 Food Pretreatment for Removal of Membrane Foulants

Despite the development of rapid detection methods (such as ELISA andPCR), reduction and/or elimination of cultural enrichment remains anessential goal to attend the need for truly real time detection methods.While detection may be measured in hours, the steps leading up todetection add days to the whole process. Therefore, there is anessential role for a time effective bacterial concentration andpurification from food sample matrices as a step preceding detection(known as pre-analytical sample processing) (Dwivedi, H. P. and Jaykus,L-A. 2011. Critical Reviews in Microbiology 37(1):40-63) (Kim, H.-Y.,Park, H.-J., and Ko, G. 2010. Journal of Food Protection72(12):2547-2552).

Microfiltration techniques represent one conceptually simple way toreduce large samples to a small volume and effectively increase cellconcentration and recovery from food without lengthy culturing andenrichment steps. However, fouling of membrane is a challenging thatneeds to be addressed in order to make this process effective. Foulingis caused by the accumulation of fats, oils, particles, and proteins onthe surface of the membrane, in a manner that causes a layer to formthat blocks transmission of fluid through the membrane (Kimura, K.,Hane, Y., Watanaeb, Y., Amy, G., Ohkuma, N. 2004. Water Research38:3431-3441) (Ladisch, M. R. 2001. Bioseparations Engineering:Principles, Practice, and Economics, 17-19, 36-47, 53-106,Wiley-Interscience, NY) (Peterkin, P. I., Sharpe, A. N. 1980. ApplEnviron Microbiol. 39(6):1138-1143).

In addition to the need of developing methods to reduce (or eliminate)membrane fouling, the understanding of bacterial surface adhesion to thesubstrate of interest (possibly contaminated food, in this case poultrymeat) is also important. Goulter et al. (Goulter, R. M, Gentle, I. R,and Dykes, G. A. L. 2009. Appl. Microbiol. 49: 1-7) and Faille et al(Faille, C., Jullien, C, Fontanine, F., Bellon-Fontanine, M., Slomianny,C., and Benezech, T. 2002. Can. J. Microbiol. 48:728-738) havepreviously described hypothesis for bacterial binding to abiotic andmetallic surfaces. Goulter suggested that bacterial bindings occurs viaa two-step mechanism: (1) bacteria attaches to a surface with aninitially reversible state which involves van der Waals forces,electrostatics, and hydrophobic/hydrophilic interactions; (2)irreversible attachment which can involve covalent bonding, hydrogenbonding, and very strong hydrophobic interactions. Faille suggested thatcell adhesion exists through a three step process: (1) physiochemicalinteractions (similar as before); (2) adhesion reinforcement throughpolymer excretion; (3) cellular division. Faille also suggests acorrelation between the micro-scale roughness of a sample and itsadhesion ability.

An embodiment of the invention addresses both aspects through a methodfor food pretreatment prior filtration using hollow fiber membranes. Inthis method, the possibly contaminated sample (for example a piece ofpoultry meat) is mixed gently with water (e.g. in a weight/volume ratioof 1:9), bringing the pH to ˜6.0, and then homogenized in a STOMACHERbag, desirably at a relatively low speed (e.g. 100 rpm) for a suitableperiod of time, for example 30 seconds. The use of lower speed is toavoid additional stress to the microorganisms, as well as the release ofmore proteins, which has been found to be one of the major membranefoulants.

The resultant preparation is then mixed for 1 hour at 37° C. and 200 rpmwith an enzyme (0.5% (v/v)) effective in hydrolyzing proteins atsubstantially neutral pH, for example the enzyme PROTEXTm 7 L, producedby Genencor International, Inc., Rochester, N.Y., USA. This is abacterial endopeptidase preparation derived from a controlledfermentation of a non-genetically modified strain of Bacillusamyloliquefaciens. In order to remove lipids, another identified majorfoulant, the enzyme-treated preparation is then passed through amembrane (for example a 2.7 micrometer glass microfiber membrane (GF/Dmicro filter)), VWR, Batavia, Ill., USA) as a pre-filtration step. Thesample is then ready to filtration by the use of hollow fiber membranes,e.g. in system 30 described in this application, for concentration andrecovery of microorganisms.

The combination of pre-filtration using glass microfiber membranes andenzyme treatment prior concentration and recovery of microorganisms fromthe food sample (e.g. poultry meat) significantly reduces the necessarytime (preferably 1-2 hours, compared to days when using standardmethods) for the whole process and allows re-use of the hollow fibermembranes if appropriated sterilization procedures are taken.

Using the developed food pretreatment method disclosed herein, theresults indicated that, although more challenging, efficient use ofhollow fiber membranes targeting bacteria in food, previouslyhomogenized before cell concentration and recovery, can be achieved in asignificantly shorter time compared to standard methods (days) by theapproach using few steps that can be integrated in one system for thewhole process of microorganism concentration and recovery fromcontaminated food.

Example 2 Rapid Detection of Pathogens in Real Time

In the developed method described herein, initially a sample ofbacterial cells is prepared from a liquid food extract using anautomatically controlled microorganism concentration instrument. Theoperation of this instrument uses the simple principle ofmicrofiltration in a continuous flowthrough type operation to decreasethe volume of the sample to a small volume, having concentration ofbacterial cells at sufficient number, so that they may be effectivelyprobed for presence of pathogen on a “first alert” basis by the use offluorescent antibodies, and detecting them using a spectrometer (forexample a USB4000-UV-VIS Miniature Fiber Optic Spectrometer).

Preliminary tests have been done with E. coli GFP (sgGFPTM: SuperGlo™Green Fluorescent Protein: a single excitation peak at 474 nm, andemission at 509 nm). Further tests for optimization include the use offluorescent micro-particles (7.-7.9 m, Spherotec, Inc., Lake Forest,Ill., USA) available with functional groups for covalent binding ofantigens or antibodies. After these initial tests for optimization, thetechnology will be applied to test first the presence of Salmonella spp.artificially spiked in food at different concentration levels. Thesamples containing Salmonella will be incubated with fluorescentantibodies for 30-60 min at room temperature before applied to cellconcentration and recovery in a system such as system 30 describedherein (less than 10 min). The preparation and use of fluorescentantibodies for detection of Salmonella in food products was taught byHaglund et al. in 1964 (Haglund J. R., Ayres J. C., Paton A. L, Kraft A.A, and Quinn L. Y. 1964. Applied Microbiology, 12(5):447-450). The useof fluorescently-labeled monoclonal antibodies, with detection bymulti-parameter flow cytometry for the rapid detection of Salmonellas inpure cultures has also been demonstrated even in the presence of highlevels of other bacterial species that would otherwise have interferedwith the results. With combinations of different antibodies, each with aunique fluorescent label, simultaneous analysis for two species ispossible (McClelland R. G. and Pinder A. C. 1994. Journal of AppliedMicrobiology, 77 (4):440-447). The method of this and other embodimentsof the invention can use anti-Salmonella fluorescent antibodiesavailable commercially.

Increased interest has been developed for rapid detection of bacteria infood, biosafety and medicinal analysis, including the identification ofinfectious agents and the rapid detection of bio-warfare agents (Tauxe,R. V. Int. J. Food Microbiol. 78. 2002. PII S0168-1605(0102)00232-00235)(Love, T. E., Jones B., Principles of Bacterial Detection: Biosensors,Recognition Receptors and Microsystems, Springer, 2008, pp. 3-13)(Zhang, J. I., Talaty, N., Costa, A. B., Xia, Y., Tao, A., Bell, R.,Callahan, J. H. and Cooks, R. G. 2011. International Journal of MassSpectometry 301:37-44). Although traditional methods such as nucleicacid hybridization (McKillip, J. L. and Drake, M. 2004. J. Food Prot.67:823-832), polymerase chain reaction (PCR) (McKillip, J. L. and Drake,M. 2004. J. Food Prot. 67:823-832) (Van Kessel, J. S, Karns, J. S. andPerdue, M. L. 2003. J. Food Prot. 66 1762-1767), enzyme linkedimmunosorbent assays (ELISA) (Mansfield, L. P. and Forsythe, S. J. 2000.Lett. Appl. Microbiol. 31:279-283) (Valdivieso-Garcia, A., Riche, E.,Abubakar, O., Waddell, T. E. and Brooks, B. W. 2001. J. Food Prot.64:1166-1171) immunofluorescence assays (IFAS) (Ng, S. P., Tsui, C. O.,Roberts, D., Chau, P. Y., Ng, M. H. 1996. Appl. Environ. Microbiol.62:2294-2302) or chemotaxonomic fingerprint by chromatographic (Huys,G., Altwegg, M., Hanninen, M. L., Vancanneyt, M., Vauterin, L., CoopmanR., Torck, U., LuthyHottenstein, J., Janssen, P. and Kersters, K. 1996.Syst. Appl. Microbiol. 19:616-623) and methods (Busse, H. J., Denner, E.B. M. and Lubitz, W. 1996. J. Biotechnol. 47 (1996) 3-38) candifferentiate bacteria at the species and sub-species level reliably,they may require hours to days to perform (Zhang, J. I., Talaty, N.,Costa, A. B., Xia, Y., Tao, A., Bell, R., Callahan, J. H. and Cooks, R.G. 2011. International Journal of Mass Spectometry 301:37-44). Thedetection method of the invention differs from those methods, first bybeing integrated to an instrument such as system 30 disclosed herein forcell concentration and recovery, and second by probing targetedpathogens in real or near real time.

Example 3 Integrated Platform for Simultaneous Concentration andRecovery of Microbial Cells

The technology integrated into a cell concentration and recovery (CCR)instrument providing system 30 herein can include easy to operatecomponents: peristaltic pumps, low (or “zero”) dead volume tubing,automated valves, sterilizable fittings, sample and solvent reservoirs,and hollow fiber microfiltration membrane module packaged with reusablehousings.

The food sample can be processed by microfiltration in a cross-flowoperation mode to concentrate the sample to a small volume, increasingthe number of microbial cells to a detectable level so that they may beeffectively probed for the presence of pathogen. The running of thisinstrument includes two modes, i.e., the cell concentration or CC mode,and the cell recovery or CR mode. Under CC mode, the sample is feed tothe membrane module with a pump. The permeate that passes the membranegoes to the permeate vessel, while the particles that are rejected bythe membrane are concentrated in the retentate. The retentate iscirculated back to the sample reservoir and further concentrated untilno or very little sample is left in the sample reservoir. A second pumpis used to pump a liquid (e.g. sterile water) flow against the hollowfiber membranes from the permeate side, e.g. to achieve a substantiallyconstant transmembrane pressure. Under the CR mode, the retentate sampleis pumped to the collection tube which can be facilitated by a two-valvethat can selectively circulate retentate back to the sample reservoir orto the collection tube.

A simplified software interface compatible with the most recent versionof LabVIEW with graphical programming for measurement and automation hasbeen developed using simulation software and an interface. The programand interface are installed in a lab laptop for displaying the pressureand flow data and controlling the unit's operation. A cycle of sampleconcentration, sample recovery, and chemical asepsis (e.g as discussedin conjunction with FIG. 1 herein) is carried out automatically with thecontrol of these operations provided through the interface.

This integrated instrument can be used to concentrate and recoverymicrobial cells from large volume of food suspensions/extracts in atime-efficient manner. The concentration and recovery of microbial cellsfrom food matrices is the basis of rapid food borne pathogen detection.

In the developed instrument, cell concentration and recovery areintegrated. The microbial cells are not only separated from the permeatebut also recovered for further analysis. This embodiment is highlyautomated and the manual handling can be minimized. The recoveredcell-containing retentate sample can be processed for subsequentpathogen detection.

Example 4 Method for Ensuring Instrument Viability

Hollow fiber (HF) ultrafiltration has been used as a potential approachto simultaneously concentrate different microorganisms in water, andreduce clogging problems compared to conventional ultrafilters(Morales-Morales H. A., Vidal G., Olszewski J., Rock C. M., Dasgupta D.,Oshima K. H. and Smith G. B. 2003. Appl. Environ. Microbiol. 69(7):4098-4102) (Hill V. R., Polaczyk A. L., Hahn D., Narayanan J., CromeansT. L., Roberts, J. M., and Amburgey J. E. 2005. Appl. Environ.Microbiol. 71(11): 6878-6884) (Smith C. M. and Hill V. R. 2009. Dead-endhollow-fiber ultrafiltration for recovery of diverse microbes fromwater. Appl. Environ. Microbiol. 75(16): 5284-5289) (Kim H.-Y., ParkH.-J., and Ko G. 2010. Journal of Food Protection 72(12): 2547-2552).Its use may offer advantages, such as facilitating the detection ofmultiple bacterial strains, removal of matrix-associated reactioninhibitors and provision of adequate sample size reductions to allow forthe use of representative food sample sizes (Stevens, K. A. Jaykus, L.A. 2004. Critical Reviews Microbiology. 30: 7-24). Recent literature hasindicated the potential efficient use of HF membranes for pathogenconcentration and recovery from water samples (Morales-Morales H. A.,Vidal G., Olszewski J., Rock C. M., Dasgupta D., Oshima K. H. and SmithG. B. 2003. Appl. Environ. Microbiol. 69(7): 4098-4102) (Hill V. R.,Polaczyk A. L., Hahn D., Narayanan J., Cromeans T. L., Roberts, J. M.,and Amburgey J. E. 2005. Appl. Environ. Microbiol. 71(11): 6878-6884)(Smith C. M. and Hill V. R. 2009. Dead-end hollow-fiber ultrafiltrationfor recovery of diverse microbes from water. Appl. Environ. Microbiol.75(16): 5284-5289) (Kim H.-Y., Park H.-J., and Ko G. 2010. Journal ofFood Protection 72(12): 2547-2552) (Stevens, K. A. Jaykus, L. A. 2004.Critical Reviews Microbiology. 30: 7-24) (Park H., Kim M. and Ko G.2010. Journal of Food Protection 73(9): 1651-1657). Recent resultsindicated that, although more challenging, by using a developedinstrument based on the use of hollow fiber membrane modules, targetedpathogens in food can be effectively concentrated and recovered forfurther identification.

In order to allow instrument re-use, and consequently enhance theeconomics for the whole process, we developed a method for cleaning andsterilization of a hollow fiber membrane instrument (e.g. providingsystem 30 described herein) for cell concentration and recovery.

Positive results has been systematically achieved by sequential use ofNaOH (0.2 M, 10 min), H₂O (5 min) to clean any trace of caustics in thesystem, 70% (v/v) ethanol (10 min) to return the system back to asterile state, and finally water one more time at the same pressure andflow rate for 5 min to clean any trace of ethanol and rehydrate themembrane. This method has been consistently used without any negativeimpact on any of the instrument parts.

The effectiveness of cleaning and sterilization has been accessedthrough microscopy analysis and plating in non- and selective media formonitoring. Results indicate the possible re-use of the hollow fibermembranes for cells concentration from food for at least about 10-20times (depending on the volume applied, and still under optimization).These tests have been done with different new hollow fiber membranemodules.

The disinfection of the whole instrument for concentration and recoveryof microorganisms before and after each filtration is important takingin consideration that bacteria have the potential for rapid reproductionunder diverse environmental conditions. The disinfection methoddeveloped and described herein allows for multiple re-use of the hollowfiber membrane modules, which will also enhances economics associated tothe whole process.

Example 5 Design and Fabrication of Multi-Tubular Membrane Bundle

The rapid detection of microorganisms against a background ofnonpathogenic bacteria in contaminated food samples requires recoveringthe bacteria from colloidal matter, fats, proteins, and other componentswhich rapidly foul membranes and stop flow in a matter of minutes.Microfiltration techniques represent a conceptually simple way to reducelarge samples to a small volume and effectively increase cellconcentration without lengthy culturing and enrichment steps. Dead-endfiltration techniques using a flat-sheet membrane have proven to beeffective for concentrating microbial cells, including L. monocytogenesand Gram-negative pathogens including Salmonella for microbiologicalanalysis of water, dairy, and food products (Carroll, S. A., Carr, L.E., Mallinson, E. T., Lamichanne, C., Rice, B. E., Rollins, D. M.,Joseph, S. W. 2000. J. Food Prot. 63(3):347-353) (Chen, W. T.,Hendrickson, R. L., Huang, C. P., Sherman, D., Geng, T., Bhunia, A. K.,Ladisch, M. R. 2005. Biotechnol Bioeng. 89:263-273) (Chen, W. T.,Ladisch, M. R., Geng, T., Bhunia, A. K. 2005. AIChE. J. 51:3305-3308)(Entis, P., Lerner, I. 2000. J Food Prot. 63(3):354-363) (Ladisch, M. R.2001. Bioseparations Engineering: Principles, Practice, and Economics,17-19, 36-47, 53-106, Wiley-Interscience, NY) (Peterkin, P. I., Sharpe,A. N. 1980. Membrane filtration of dairy products for microbiologicalanalysis. Appl. Environ. Microbiol. 39(6):1138-1143) (Hill, V. R.,Polaczyk, A. L., Hahn, D., Narayanan, J., Cromeans, T. L., Roberts, J.M., and Amburgey, J. E. 2005. Appl. Environ. Microbiol.71(11):6878-6884). It has been shown that sequential filtration througha depth filter followed by a screen filter could concentrate 100 mlsamples having as few as 20 CFU/ml to a volume of 50 l with 2,000 CFU/ml(Chen, W. T., Ladisch, M. R., Geng, T., Bhunia, A. K. 2005. AIChE. J.51:3305-3308) (Entis, P., Lerner, I. 2000. J Food Prot. 63(3):354-363).However, due to fouling of the membrane and the need for removing andhandling it, an alternate method was needed. Hollow fiber (HF) membranesoffer several advantages over the traditional flat-sheet membranes: ahigh surface area to volume ratio, a higher flux per unit volume of themembrane module, and continuous operation that avoids manual handling ofthe membrane and sample. The membrane may be back-flushed to recoverconcentrated cells in an aqueous buffer or flushed with an elutionbuffer to recover cells from the device platforms for detection andidentification. Recent literature has taught certain uses of HFmembranes for pathogen concentration and recovery from water samples(Kim, H.-Y., Park, H.-J., and Ko, G. 2010. Hollow fiber ultrafiltrationfor the concentration and simultaneous recovery of multiple pathogens incontaminated foods. Journal of Food Protection 72(12):2547-2552)(Morales-Morales, H. A., Vidal, G., Olszewski, J., Rock, C. M.,Dasgupta, D., Oshima, K. H. and Smith, G. B. 2003. Appl. Environ.Microbiol. 69(7):4098-4102) (Smith, C. M. and Hill, V. R. 2009. Dead-endhollow-fiber ultrafiltration for recovery of diverse microbes fromwater. Appl. Environ. Microbiol. 75(16):5284-5289) (Fukushima, H,Katsube, K, Hata, Y, Kishi, R, Fujiwara, S. 2007. Appl. Environ.Microbiol 73(1):92-100).

In aspects of the present invention a hollow fiber ultrafiltrationmembrane module (HFM) was assembled for use in a continuous cellconcentration device (CCC), for example providing system 30 describedherein. This HFM was constructed from twelve 0.22 μm commercialpolysulfone hollow fibers available from Minntech Corporation, IDEX PEEKtubing, and Loctite Medical Device Epoxy Adhesive. Hollow fibermembranes are inserted through an assembled PEEK tubing skin and gluedat the ends with medical device epoxy. HFMs, are based size-exclusionultrafiltration. Specifically for this purpose, the HFM was designed tooperate under creep flow. The HFM also uses a second water circulationline to increase the transmembrane flux through the device bymaintaining a positive pressure on the outside of the ultrafiltrationmembrane. This ultrafiltration membrane module allows for theultrafiltration and concentration of particles (including pathogens andfood stuffs) which are larger than 0.22 micrometers in diameter. Rapidconcentration of food samples can be used for the early detection andidentification of contaminants and pathogens present in food samples.

Example 6 Spectral Analysis of Baseline Noise for On-Line Detection ofPathogens

A combination of rapid concentration of cells from washate or extractedfood matrices will be accompanied by the addition of GFP (GreenFluorescent Protein) linked to antibodies specific for proteinsdisplayed by pathogenic cells. The principle of detection is based onthe binding of a polyclonal or monoclonal antibody to the surface of acell, causing the cell to anchor a chromophore (GFP) to the surface.Consequently, as the cell becomes more concentrated the bound antibodywill become visible as specks of light, associated with specificparticles (i.e. cells). In order to achieve detection, however, theconcentration of pathogenic cells would needs to be relatively high(e.g. beneficially 1,000 to 10,000 cells per ml), and therefore anindirect measurement will be needed for this approach to be practicaland economical.

The method of this embodiment concentrates the cells, and then injectsan amount (typically small) of monoclonal antibody with the GFP attachedso that it binds to the pathogenic cells, which are in the concentratebeing re-circulated through the hollow fiber membrane system (e.g.system 30 described herein). Initially, the GFP would cause a verystrong response (such as would be obtained using the spectrometer fromOcean Optics for detection, when applying green fluorescent protein toit), and consequently, reliable detection of the pathogenic cells wouldnot be possible. However, as the liquid from the sample being processedis washed through the membrane of the hollow fibers (becoming permeate),cells to which the GFP would be attached (pathogenic cells) would beretained. Consequently, as a background, due to the presence of solubleGFP-bound monoclonal antibodies, is reduced through membranemicrofiltration, the signal from cells attached to the monoclonalantibodies would then become more prevalent.

Since the number of pathogenic cells is small, a measure that can bebeneficially used for their detection is an increase in baseline noise.The detection is noisy, particularly at higher sensitivities. Thus, ifcells are present to which the monoclonal antibody with GFP attached areconcentrated, the signal would become more noisy as GFP bound to cellspasses through the detector. Consequently, an online spectral analysisof this phenomenon would indicate rather random noise is becoming lessrandom, and thereby indicate the potential presence of pathogenic cellsagainst a background of a small amount of GFP, and a large number ofnon-pathogenic cells. Thus, a combination of cell concentration,measured and timed addition of amounts of monoclonal antibodies to whichGFP is attached been reacted, binding of the antibody to pathogeniccells, coupled with simultaneous washing out of monoclonal antibody withGFP (in free solution) through the action of the hollow fiber membranedevice may be used to enable rapid and on-line detection of pathogeniccells. Since the number of pathogenic cells, if present, would likely besmall, a change in the signal-to-noise-ratio and the frequency of spikesin the noise, would be used as a first indicator of the presence ofpathogenic cells.

Mathematically, this would be achieved in real time using spectralanalysis, coupled with a mini-GFP detector, such as the Ocean OpticsSystem, that is being used on the concentration device. An embodiment ofthe invention thus includes: 1) Use of a concentration device to rapidlyincrease the total number of living microbial cells; 2) measured andtimed addition of small amounts of monoclonal antibody, to which GFP isattached, during times during which cell concentration is occurring; 3)simultaneous removal of soluble monoclonal antibody/GFP through thewashing action of the cell concentration step (additional buffer may beadded) with specific binding of the antibodies (green fluorescing orother fluorescing to pathogens) occurring at the same time; and 4)detection of cells to which the fluorescent antibody has bound throughchanges in baseline noise. This can give a first indicator of thepotential presence of pathogenic cells, i.e. first alert. If pathogeniccells are found, the sample could then be further processed in order togive a more thorough analysis, using ELISA, or PCR-type analysis, or anyother suitable analysis. In addition, the pattern of subsequent growthin culture may be measured using a laser light optical system (such asBardot), or using Raman spectroscopy for detecting large amounts ofpathogenic cells, as a second analysis.

Example 7 Computer Interface for Hands-Off Control of PathogenConcentration and Recovery System

An interface for hands-off concentration, recovery, and aseptic clean-upof a rapid system for concentrating and recovering living cells in foodmatrices is described. The interface enables hands-off control andmonitoring of a system in which fluid is re-circulated through amembrane, which allows some of the fluid to pass. The resulting solidsinclude microbial cells, which are concentrated, and then re-circulateduntil the desired concentration is attained. The system (e.g. system 30as described herein) has various monitors including pressure, flow rate,and mass, as well as an on-line spectrophotometer for detectingfluorescent protein, which may be attached to pathogenic cells. Inaddition, the system includes a controller with executable softwarewhich communicates with the pumps and the valves on the instrument (e.g.system 30), is capable of detecting leaks so that fluid, which maycontain pathogens, upon which the fluid is automatically diverted intocaustic or bleach or another sterilization agent to kill themicroorganisms. In addition, the system can have programmed into it anautomated sequence for cleaning of the membranes, aseptic processing ofthe system so that ethanol or other fluids for killing microorganismsbetween samples is circulated, followed by a clean-up step, in whichcleaning solutions are removed from the system so that it may be usedagain. This hands-off system enables the re-use of the membranes, forexample for up to 10-20 times and thereby enables a more economicoperation of a cell concentration and recovery system.

This embodiment involves the manner and sequence in which theconcentration, recovery, wash, cleaning, and equilibration steps arecarried out; and the controller (e.g. computer) with executable softwareor other programming, which enables operation of the system in ahands-off and safe manner, and so that detection of leaks would resultin diversion of the fluid into a solution which would kill anymicroorganisms, whether they are pathogenic or not. This particularsystem embodiment also enables the cell concentration recoveryinstrument to be operated such that rapid concentration and recovery, aswell as first alert detection of the cells, is possible.

Example 8 Constant Flux Concentration of Microorganisms

Membrane filtration of microorganisms from extracts from food matricesare hindered by fouling. This must be overcome in order to achieve rapidrecovery and concentration of microorganisms derived from extracts offood matrices, so that the concentrated microorganisms may be probed forthe presence of pathogens. Reliable detection of foodborne pathogens isan integral part of food safety and that current detection of foodpathogens is slow in large part due to the time required to amplifymicrobial cultures derived from foods, followed by selective culture toincrease the numbers of pathogenic microorganisms if they are present.

A faster detection scheme is desirable, since the detection of pathogenstakes place before foods are distributed to the consumers. Alab-on-a-chip is used for rapid detection using either capture byselective antibodies, and/or on-chip PCR to identify cells that may havebeen captured (e.g. in a retentate sample produced by system 30) withrespect to whether they are pathogens. In order to achieve rapiddetection, concentrated cells must be presented to the chip so thatdetection can proceed with reasonable accuracy. The volumes on suchchips are typically on the order of 100 nL, with 1 cell/100 nL's beingequivalent to 10,000 cells/mL, or 10⁷ cells/L. Consequently, for thepresence of pathogens to be detected at concentrations from one cell to1,000 cells a volume of 100 mL's or more must be processed into a finalvolume of 1 mL or less. Consequently, large volumes of fluid containingpotential pathogens, together with a large background of non-pathogenicmicroorganisms, must be amplified. The preferred approach is presentedherein, in which the cells are concentrated preferably to a volume ofless than 500 microliters, giving cell concentrations which may forexample be 100-1,000 cells/ml. When preceded by or followed by growth ofthe microbial cells for a period of several hours, detectableconcentrations can result.

This embodiment can use simultaneous flow on both retentate and permeateside of a hollow fiber (e.g. system 30) or equivalent membrane system.The flow of fluid through the membrane into a moving fluid on thepermeate side keeps the pressure drop across the membrane at a constantor substantially constant difference, while enabling a rapid flow rateacross the inner side of the membrane (retentate side) to help minimizefouling. Fouling by cake formation will be negligible when shear induceddiffusion is greater than actual convection. Small particles can foulthe membrane, because shear gradients affect them less (these particlesare larger than smaller colloidal matter or large molecules, and areless mobile).

Fouling may be non-uniform along the length of the membrane. Sinceactual convection and shear diffusion are directly coupled under normaloperation, the flux, and therefore tangential convection, isproportional to transmembrane pressure. As fluid flows along the lengthof the membrane, there will be pressure loss per unit length of themembrane. The actual convection will start at a high rate and thendecrease along the axis of the membrane. At the same time, the magnitudeof shear-induced diffusion remains constant. Ideally, the shear gradientwill be high and the tangential connection will remain low. Under normaloperations, this is not achieved because tangential convection andshear-induced diffusion are coupled.

To address these issues, this embodiment can include pumping apermeate-side liquid (e.g. water or a buffer solution) across thepermeate side of the hollow fiber or other membrane such that thetransmembrane pressure will remain constant along the length of themembrane. This will uncouple the forward and backward transportmechanisms, thereby allowing for increased membrane performance. A flowof buffer, which has a much lower concentration of dissolved solutesthan typically occurs on the retentate side, as well as a lowerviscosity, when pumped on the permeate side of the membrane, has beenfound to enable enhanced flux and reduced fouling for a solutioncontaining extracted molecules and particles, as well as microorganisms.Operating according to this embodiment of the invention has been foundto maintain flux and enables rapid concentration even when infant foodformula (which has many fat molecules and other species that rapidlyfoul the membrane) is used on the retentate side of the membrane.

Example 9 Enzyme-Assisted Pathogen Detection

Various materials that are used in the production of foods, and thepreparation of baked goods, may contain microbial pathogens. These foodsmight include flour, condiments, spices, chocolate, and related drygoods. These materials may contain microorganisms, and in order for themto be probed for possible presence of pathogens, the food material mustsomehow be altered so that any microorganisms present would be releasedinto a liquid solution. Once the liquid solution is obtained, themicroorganisms may be rapidly concentrated and recovered using amembrane-based concentration instrument. Foods that may harbor bacteriaor pathogens include dry cereals (such as corn flakes, Cheerios, andother dry breakfast cereals), as well as numerous confectionary productssuch as cookies, cakes, and crackers. The embodiment herein includes theutilization of a combination of cellulases, hemicellulases, xylanases,amylases, glucosidases, proteases, lipases, nucleases, and/or esterases,formulated in a manner that they can be added to relatively smallsamples of food and liquefy these foods by hydrolyzing carbohydrates,proteins, and lipids into their monomeric components, thereby allowingthe food material to dissolve. Once this occurs, microorganisms that arecontained in these foods are released, and may be filtered andconcentrated using the membrane-based device described in the presentapplication and embodiments thereof.

The types of enzymes, and their formulation will depend on the type offood being processed into a sample for probing for the presence ofpathogenic microorganisms and therefore will lead to a number ofdifferent enzyme formulations. It is also possible that additives,including small amounts of detergents, salts, buffer, and proteins (inaddition to enzymes) will enhance the rate and extent of the release ofmicroorganism that may be present in the food.

The invention, coupled with methods for rapid concentration anddetection of cells (for example as described with FIG. 1 and/or usingsystem 30 described in conjunction with FIG. 2), enables probing of drygoods and various types of food materials that are used in bakeryproducts and dry breakfast cereals to be rapidly probed for the presenceof bacteria, and for pathogens that may exist in the microbialpopulation (if present). Since the microorganisms are internal to thefood particles (i.e., “baked- or extruded-in”) these microorganismswould have to be released from the solid particles. In order to do this,and to make the material amenable to rapid processing and detection, thenon-microbial components need to be dissolved in conditions which aremild and which preserve the viability of the microorganisms.

Consequently, combinations of enzymes are used which will break downthese food matrices for the purpose of generating either solublesolutions, or suspensions of very fine particles of these food materialsso that they might be rapidly filtered and ultimately concentrated usinga novel rapid concentration and recovery system, e.g. system 30described herein. The use of enzymes, including at high concentrations,is within the realm of economic viability for the processing of foodsamples so that presence of pathogens might be detected.

This approach may also be applied to vegetables (in which microorganismsare taken up by the vascular tissue, and therefore, can only be probedor detected if the vascular tissue is dissolved (i.e. hydrolyzed away).At the same time, it has been shown that a subset of these enzymes maybe used in order to assist in the processing of extracts from chickenand that these microorganisms do not have negative effect on themicroorganisms present in these food samples. At a high level, thisembodiment involves a formulation of enzymes that may be added to foodmaterials preferably at room conditions in order to quickly dissolvethese materials within 30 minutes so that they may be filtered, and theliquid passed through a membrane-based filtration and concentrationsystem (e.g. system 30 described herein). This method embodiment mayhave possible applications to other approaches to rapid pathogendetection in which the microorganisms are suspended would be eitherplated out, subjected to imaging, for ELISA assays, PCR, labeling withbiomarkers, and use of microscopy. Since the food particles wouldrepresent a background which must be removed for analyses to be carriedout, and since the food particles may, in fact, encapsulate or enclosethe microorganisms, the use of enzyme will greatly aid the release ofmicroorganisms so they may be tested for the presence of pathogens.

Example 10 Concentration and Recovery of Viable Microbial Cells

After cells are concentrated using an instrument to process food sampleby microfiltration in a cross-flow operation to a small volume (e.g.using system 30 described herein), and consequently increasing thenumber of microbial cells to a detectable level, the cells need to beefficiently recovered in order to be effectively probed for the presenceof possible pathogens in food.

This embodiment provides a method to recovery the microbial cells thathave been concentrated in the retentate after microfiltration. Arecovery buffer is used. It is prepared for example by dissolving anonionic surfactant such as tween 20 in sterile deionized water at thedilution of 0.001% (v/v). Tween 20, also known generically asPolysorbate 20, is a surfactant and spreading agent that lower thesurface tension of a liquid, the interfacial tension between twoliquids, or that between a liquid and a solid, and thus can be used toremove the microbial cells attached to the membrane surface. Othersuitable surfactants for these purposes may also be utilized. Thestability and relative non-toxicity of tween 20 allows it to bebeneficially used in the recovery of microbial cells. The dosage oftween 20 is as low as 0.001% (v/v) for the reason that highconcentrations of tween may produce a large amount of foam which isundesirable for the performance of microfiltration (e.g. in system 30).The dose of the recovery buffer used in the cell recovery step can belower than the initial food sample volume, for example 10 ml of bufferper 250 ml of initial sample loading. After the microfiltration of thesample containing microbial cells, the recovery buffer is processedthrough the microfiltration system (e.g. system 30) under the same ordifferent conditions until it is collected with the prior-collected cellretentate sample.

The method can be applied in the recovery/harvest of microbial cellsconcentrated in the retentate after microfiltration, such as describedin connection with FIG. 1 and/or in system 30 of FIG. 2. This methodprovides significant and consistent results for concentration andrecovery of microbial cells (and therefore provide enough cells forprobing for the presence of pathogens) from many types of food matrices.

Example 11 Concentration and Recovery of Viable Microbial Cells

After cells are concentrated using an instrument to process food sampleby microfiltration in a cross-flow operation to a small volume, andconsequently increasing the number of microbial cells to a detectablelevel, the cells need to be efficiently recovered in order to beeffectively probed for the presence of possible pathogens in food.

Developed is a method to recovery the microbial cells that have beenconcentrated in the retentate after microfiltration. The recovery bufferis prepared by dissolving tween 20 in sterile deionized water at thedilution of 0.001% (v/v). Tween 20, also known generically asPolysorbate 20 is a surfactant and spreading agent that lower thesurface tension of a liquid, the interfacial tension between twoliquids, or that between a liquid and a solid, and thus can be used toremove the microbial cells attached to the membrane surface. Itsstability and relative non-toxicity allows it to be used in the recoveryof microbial cells. The dosage of tween 20 is as low as 0.001% (v/v) forthe reason that results indicate that higher concentrations of tween mayproduce large amount of foam which is undesirable for the performance ofmicrofiltration. The dose of the recovery buffer is 10 ml per 250 mlinitial sample loading. After the microfiltration of the samplecontaining microbial cells, the recovery buffer is processed by themicrofiltration system under the same conditions until finished.

The method can be applied in the recovery/harvest of microbial cellsconcentrated in the retentate after microfiltration. This method isessential to get significant and consistent results for concentrationand recovery of microbial cells (and therefore provide enough cells forprobing to the presence of pathogens) from many types of food matrices.

The results in literature for pathogens recovered from water indicatedrecoveries varying from ˜40-95% depending on the experimental conditionsused samples (Kim, H.-Y., Park, H.-J., and Ko, G. 2010. Hollow fiberultrafiltration for the concentration and simultaneous recovery ofmultiple pathogens in contaminated foods. Journal of Food Protection72(12):2547-2552) (Smith, C. M. and Hill, V. R. 2009. Dead-endhollow-fiber ultrafiltration for recovery of diverse microbes fromwater. Appl. Environ. Microbiol. 75(16):5284-5289) (Park H., Kim M. andKo, G. 2010. Journal of Food Protection 73(9):1651-1657). Cells fromnatural flora and artificially spiked Salmonella (102-103 CFU/mL) cellspresent in a more complex sample matrix (chicken rinse) varying from60-80%, depending on the experimental conditions used have beenrecovered cells.

SPECIFIC LISTED EMBODIMENTS

The disclosure herein includes the following embodiments:

Embodiment 1

A method for processing a food sample, comprising:

-   -   preparing a liquefied extract of a food sample; and    -   processing the liquefied extract by hollow fiber membrane        filtration to form a concentrate of cells from the liquefied        extract.

Embodiment 2

The method of Embodiment 1, wherein said preparing includesenzymatically digesting the food sample.

Embodiment 3

The method of Embodiment 1 or 2, wherein the hollow fiber membranefiltration is conducted with a cross flow pattern.

Embodiment 4

The method of any preceding Embodiment, wherein the concentrate of cellsincludes pathogenic cells.

Embodiment 5

The method of any preceding Embodiment, also comprising assessing theconcentrate of cells for the presence of pathogenic cells.

Embodiment 6

A method for preparing a liquefied extract of a food sample, comprising:

-   -   homogenizing a solid food sample under controlled shear        conditions to form a homogenized food sample substantially        retaining a viable cellular content of the solid food sample;    -   enzymatically digesting the homogenized food sample to hydrolyze        proteins in the homogenized food sample and form a digested food        sample; and    -   processing the digested food sample to remove lipids from the        digested food sample, to form a lipid-depleted food sample.

Embodiment 7

The method of Embodiment 6, wherein said processing includes filtering.

Embodiment 8

The method of Embodiment 7, wherein the filtering includes filteringwith a glass fiber membrane

Embodiment 9

The method of any one of Embodiments 6 to 8, also comprisingconcentrating the lipid-depleted food sample in cells.

Embodiment 10

The method of Embodiment 9, wherein said concentrating comprisesfiltering the lipid-depleted food sample with a hollow fiber membranefilter.

Embodiment 11

The method of Embodiment 10, wherein said filtering is conducted undercross flow conditions.

Embodiment 12

A method for testing for pathogenic cells, comprising:

-   -   contacting a viable cellular concentrate from a food sample with        a fluorescent antibody dye that specifically binds to the        pathogenic cells, to form a contacted sample;

processing the contacted sample under conditions to remove unboundamounts of the fluorescent antibody dye, to form a dye-depleted sample;and

-   -   spectroscopically interrogating the dye-depleted sample for the        fluorescent antibody dye.

Embodiment 13

The method of Embodiment 12, wherein the viable cellular concentrate isprepared by a process including:

homogenizing a solid food sample under controlled shear conditions toform a homogenized food sample substantially retaining a viable cellularcontent of the solid food sample;

-   -   enzymatically digesting the homogenized food sample to hydrolyze        proteins in the homogenized food sample and form a digested food        sample;    -   processing the digested food sample to remove lipids from the        digested food sample, to form a lipid-depleted food sample; and

concentrating the lipid-depleted food sample in cells.

Embodiment 14

The method of Embodiment 13, wherein said concentrating comprisesfiltering the lipid-depleted food sample with a hollow fiber membranefilter.

Embodiment 15

The method of Embodiment 14, wherein said filtering is conducted undercross flow conditions.

Embodiment 16

The method of any of Embodiments 12 to 15, wherein saidspectroscopically interrogating is conducted while the lipid-depletedfood sample is flowing in a conduit.

Embodiment 17

The method of any one of Embodiments 12 to 16, wherein said contacting,processing, and spectroscopically interrogating are each conductedmultiple times.

Embodiment 18

A method for concentrating and recovering cells from a food sample,comprising:

-   -   processing the liquefied extract by hollow fiber membrane        filtration to form a liquid concentrate of cells from the        liquefied extract; and

recovering the liquid concentrate of cells.

Embodiment 19

The method of Embodiment 18, wherein said recovering comprises passingan aqueous surfactant solution through the hollow fiber membrane.

Embodiment 20

The method of Embodiment 19, wherein the aqueous surfactant solution hasa surfactant concentration of less than about 0.01% volume/volume.

Embodiment 21

The method of Embodiment 19 or 20, wherein the surfactant is apolysorbate.

Embodiment 22

The method of Embodiment 21, wherein the surfactant is polysorbate 20.

Embodiment 23

A method for processing a hollow fiber membrane that has contacted aliquefied food sample, comprising:

(i) passing a sodium hydroxide solution through the hollow fibermembrane;

(ii) passing an aqueous medium through the hollow fiber membrane afterstep (i) to rinse sodium hydroxide residues from said membrane;

(iii) passing a sterilant solution through the hollow fiber membraneafter step (ii); and

(iv) passing an aqueous rinse medium through the hollow fiber membraneafter step (iii).

Embodiment 24

The method of Embodiment 23, wherein the aqueous medium in step (i)and/or step (iv) is water.

Embodiment 25

The method of Embodiment 23 or 24, wherein the sterilant solutioncomprises an alcohol.

Embodiment 26

The method of Embodiment 25, wherein the alcohol is ethanol.

Embodiment 27

A method for concentrating and recovering cells from a food sample,comprising:

(i) passing a liquefied extract of the food sample through the interiorof a hollow fiber membrane to form a permeate exterior of the hollowfiber membrane and a liquid concentrate of cells in the interior of thehollow fiber membrane; and

(ii) passing an aqueous solution against an exterior surface of thehollow fiber membrane during said passing step (i).

Embodiment 28

The method of Embodiment 27, wherein said passing step (ii) comprisespassing the aqueous solution co-current to a flow of the liquefiedextract through the interior of the hollow fiber membrane.

Embodiment 29

The method of Embodiment 27 or 28, also comprising preparing theliquefied extract by a process including enzymatic digestion of the foodsample.

Embodiment 30

A method for assessing a solid food sample for the presence ofpathogenic cells, comprising:

(i) suspending the solid food sample in an aqueous medium to form asuspended sample medium;

(ii) homogenizing the suspended sample medium under controlled shearconditions to form a homogenized food sample, said homogenizingconducted under conditions effective to substantially retain any viablepathogenic cells from the solid food sample;

(iii) enzymatically digesting the homogenized food sample to hydrolyzeproteins in the homogenized food sample and form a digested food samplemedium;

(iv) processing the digested food sample medium to remove lipids andform a lipid-depleted food sample extract liquid;

(v) passing the lipid-depleted food sample extract liquid through theinterior of a hollow fiber membrane to form a permeate exterior of thehollow fiber membrane and a liquid concentrate containing cells in theinterior of the hollow fiber membrane; and

(vi) passing an aqueous solution against an exterior surface of thehollow fiber membrane during said passing step (v); and

-   -   (vii) assessing the liquid concentrate containing cells for the        presence of viable pathogenic cells.

Embodiment 31

The method of Embodiment 30, also comprising, after step (vii):

(viii) passing a defouling liquid through the hollow fiber membrane;

(ix) passing an aqueous rinse medium through the hollow fiber membraneto remove defouling solution residues from step (viii);

(x) passing a sterilant liquid through the hollow fiber membrane; and

(xi) passing an aqueous rinse medium through the hollow fiber membraneafter step (x) to remove sterilant liquid residues from the hollow fibermembrane.

Embodiment 32

The method of Embodiment 31, also comprising repeating steps (i) to(vii) after step (xii).

Embodiment 33

The method of any one of Embodiments 1 to 32, wherein any one, anycombination, or all of the recited steps are conducted under the controlof a controller.

Embodiment 34

The method of Embodiment 33, wherein the controller is a computer.

Embodiment 35

A processing system, configured to be capable of carrying out a methodaccording to any one of Embodiments 1 to 34.

Embodiment 36

The system of Embodiment 35, comprising respective reservoirs for anyone or all recited liquids or mediums used in the method according toany one of Embodiments 1 to 34.

Embodiment 37

The system of Embodiment 35 or 36, comprising a hollow fiber membranefiltration module.

Embodiment 38

The system of Embodiment 37, comprising a first pump for poweringcirculation of liquid on a retentate side of said module, and a secondpump for powering circulation of liquid on a permeate side of saidmodule.

Embodiment 39

The system of Embodiment 38, comprising a detector configured to detectfluorescence in-line in a liquid flowing in a path travelling throughthe retentate side of said module.

Embodiment 40

The system of Embodiment 39, comprising a source of fluorescent dyefluidly coupled to said path.

Embodiment 41

The system of Embodiment 40, wherein said fluorescent dye bindsspecifically to a target pathogenic cell.

Embodiment 42

The system of any of Embodiments 35 to 41, comprising:

a hollow fiber membrane filtration module having a retentate side and apermeate side;

a reservoir for holding an aqueous recovery buffer for removing cellsfrom membrane surfaces fluidly coupled to the retentate side;

a reservoir for holding a liquefied food sample fluidly coupled to theretentate side;

a reservoir for holding a defoulant liquid fluidly coupled to theretentate side; and

a reservoir for holding a sterilant fluid fluidly coupled to theretentate side.

Embodiment 43

The system of Embodiment 42, also comprising a reservoir for holding aliquid medium fluidly coupled to the permeate side of the filtrationmodule.

Embodiment 44

The system of any of Embodiments 37 to 43, also comprising a detectorfor detecting pathogenic cells, said detector positioned to receive aconcentrated cell sample formed on the retentate side of a hollow fiberfiltration module.

The uses of the terms “a” and “an” and “the” and similar references inthe context of describing the invention (especially in the context ofthe following claims) are to be construed to cover both the singular andthe plural, unless otherwise indicated herein or clearly contradicted bycontext. Recitation of ranges of values herein are merely intended toserve as a shorthand method of referring individually to each separatevalue falling within the range, unless otherwise indicated herein, andeach separate value is incorporated into the specification as if it wereindividually recited herein. All methods described herein can beperformed in any suitable order unless otherwise indicated herein orotherwise clearly contradicted by context. The use of any and allexamples, or exemplary language (e.g., “such as”) provided herein, isintended merely to better illuminate the invention and does not pose alimitation on the scope of the invention unless otherwise claimed. Nolanguage in the specification should be construed as indicating anynon-claimed element as essential to the practice of the invention.

While the invention has been illustrated and described in detail in thedrawings and foregoing description, the same is to be considered asillustrative and not restrictive in character, it being understood thatonly the preferred embodiment has been shown and described and that allchanges and modifications that come within the spirit of the inventionare desired to be protected. In addition, all references cited hereinare indicative of the level of skill in the art and are herebyincorporated by reference in their entirety.

1. A method for processing a food sample, comprising: preparing a liquefied extract of a food sample; and processing the liquefied extract by hollow fiber membrane filtration to form a concentrate of cells from the liquefied extract.
 2. The method of claim 1, wherein said preparing includes enzymatically digesting the food sample.
 3. The method of claim 1 or 2, wherein the hollow fiber membrane filtration is conducted with a cross flow pattern.
 4. The method of claim 1, wherein the concentrate of cells includes pathogenic cells.
 5. The method of claim 1, also comprising assessing the concentrate of cells for the presence of pathogenic cells.
 6. A method for preparing a liquefied extract of a food sample, comprising: homogenizing a solid food sample under controlled shear conditions to form a homogenized food sample substantially retaining a viable cellular content of the solid food sample; enzymatically digesting the homogenized food sample to hydrolyze proteins in the homogenized food sample and form a digested food sample; and processing the digested food sample to remove lipids from the digested food sample, to form a lipid-depleted food sample.
 7. The method of claim 6, wherein said processing includes filtering.
 8. The method of claim 7, wherein the filtering includes filtering with a glass fiber membrane 9-11. (canceled)
 12. A method for testing for pathogenic cells, comprising: contacting a viable cellular concentrate from a food sample with a fluorescent antibody dye that specifically binds to the pathogenic cells, to form a contacted sample; processing the contacted sample under conditions to remove unbound amounts of the fluorescent antibody dye, to form a dye-depleted sample; and spectroscopically interrogating the dye-depleted sample for the fluorescent antibody dye.
 13. The method of claim 12, wherein the viable cellular concentrate is prepared by a process including: homogenizing a solid food sample under controlled shear conditions to form a homogenized food sample substantially retaining a viable cellular content of the solid food sample; enzymatically digesting the homogenized food sample to hydrolyze proteins in the homogenized food sample and form a digested food sample; processing the digested food sample to remove lipids from the digested food sample, to form a lipid-depleted food sample; and concentrating the lipid-depleted food sample in cells.
 14. The method of claim 13, wherein said concentrating comprises filtering the lipid-depleted food sample with a hollow fiber membrane filter.
 15. (canceled)
 16. The method of claim 12, wherein said spectroscopically interrogating is conducted while the lipid-depleted food sample is flowing in a conduit.
 17. (canceled)
 18. A method for concentrating and recovering cells from a food sample, comprising: processing a liquefied extract of a food sample by hollow fiber membrane filtration to form a liquid concentrate of cells from the liquefied extract; and recovering the liquid concentrate of cells.
 19. The method of claim 18, wherein said recovering comprises passing an aqueous surfactant solution through the hollow fiber membrane.
 20. The method of claim 19, wherein the aqueous surfactant solution has a surfactant concentration of less than about 0.01% volume/volume.
 21. The method of claim 19 or 20, wherein the surfactant is a polysorbate.
 22. (canceled)
 23. A method for processing a hollow fiber membrane that has contacted a liquefied food sample, comprising: (i) passing a sodium hydroxide solution through the hollow fiber membrane; (ii) passing an aqueous medium through the hollow fiber membrane after step (i) to rinse sodium hydroxide residues from said membrane; (iii) passing a sterilant solution through the hollow fiber membrane after step (ii); and (iv) passing an aqueous rinse medium through the hollow fiber membrane after step (iii). 24-26. (canceled)
 27. A method for concentrating and recovering cells from a food sample, comprising: (i) passing a liquefied extract of the food sample through the interior of a hollow fiber membrane to form a permeate exterior of the hollow fiber membrane and a liquid concentrate of cells in the interior of the hollow fiber membrane; and (ii) passing an aqueous solution against an exterior surface of the hollow fiber membrane during said passing step (i).
 28. The method of claim 27, wherein said passing step (ii) comprises passing the aqueous solution co-current to a flow of the liquefied extract through the interior of the hollow fiber membrane.
 29. The method of claim 27 or 28, also comprising preparing the liquefied extract by a process including enzymatic digestion of the food sample.
 30. A method for assessing a solid food sample for the presence of pathogenic cells, comprising: (i) suspending the solid food sample in an aqueous medium to form a suspended sample medium; (ii) homogenizing the suspended sample medium under controlled shear conditions to form a homogenized food sample, said homogenizing conducted under conditions effective to substantially retain any viable pathogenic cells from the solid food sample; (iii) enzymatically digesting the homogenized food sample to hydrolyze proteins in the homogenized food sample and form a digested food sample medium; (iv) processing the digested food sample medium to remove lipids and form a lipid-depleted food sample extract liquid; (v) passing the lipid-depleted food sample extract liquid through the interior of a hollow fiber membrane to form a permeate exterior of the hollow fiber membrane and a liquid concentrate containing cells in the interior of the hollow fiber membrane; and (vi) passing an aqueous solution against an exterior surface of the hollow fiber membrane during said passing step (v); and (vii) assessing the liquid concentrate containing cells for the presence of viable pathogenic cells.
 31. The method of claim 30, also comprising, after step (vii): (viii) passing a defouling liquid through the hollow fiber membrane; (ix) passing an aqueous rinse medium through the hollow fiber membrane to remove defouling solution residues from step (viii); (x) passing a sterilant liquid through the hollow fiber membrane; and (xi) passing an aqueous rinse medium through the hollow fiber membrane after step (x) to remove sterilant liquid residues from the hollow fiber membrane. 32-44. (canceled) 